A Mirror Image Protein Antagonist of VEGF-A: Total Chemical Synthesis and Racemic Crystallography of A Heterochiral Protein Complex

Abstract :

Using a unique combination of total chemical protein synthesis and mirror image phage display, we have systematically developed a D-protein antagonist of VEGF-A function. The bottleneck in mirror-image phage display is the preparation of the D-protein form of the target molecule, which can only be achieved by chemical synthesis. We prepared the mirror-image form of VEGF-A, i.e. D-VEGF-A, from three unprotected synthetic peptide segments stitched together by one-pot native chemical ligations. Phage displayed libraries of a novel L-protein scaffold were then screened against the D-VEGF-A to identify high affinity binders. The mirror-image form of the selected L-protein binder, i.e. the corresponding D-protein binder, was then chemically synthesized and was shown to specifically bind to native VEGF165 and to inhibit receptor binding. As expected, the D-protein antagonist was found to be non-immunogenic and had a longer half-life in vivo in mice. The binding mode of the D-protein antagonist was determined by high-resolution racemic X-ray crystallography of the {VEGF–A plus D-protein antagonist} heterochiral protein complex. The detailed structural information obtained from this crystal structure will enable the development of improved D-protein antagonists of VEGF-A.